Sickle cell disease (SCD) is a common disease caused by a single amino acid substitution, ValSGIn, in the beta globin gene. Due to tremendous scientific advances over the past 60 years, we now understand the molecular pathophysiology of SCD in exquisite detail. Unfortunately, progress in the development of novel therapies has been slower. Prospects for an effective therapy appear promising as extensive evidence demonstrates that reactivation of fetal hemoglobin prevents polymerization of deoxygenated hemoglobin, the central abnormality in SCD. In the past, a high throughput screen for multiple transcripts in each well of a multiwell plate has not been feasible. We have developed an assay that detects seven distinct globin transcripts and GAPDH expression in each well of a multiwell plate with high fidelity and at low cost, enabling us to screen libraries of small molecules in high throughput for compounds that differentially regulate expression of the globin genes. We have designed our assay to analyze expression of globin genes in primary human erythroid progenitor cells cultured in vitro. RNA is captured on oligo-dT coated 384-well plates (RNAture) and reverse transcribed. The cDNA for each gene of interest is amplified by ligation mediated PCR, labeled amplicons are captured on fluorescent microspheres (Luminex), and the relative abundance of different amplicons is detected by high speed flow cytometry. The entire process of RNA purification, ligation mediated amplification, fluorescent bead detection, and data acquisition is amenable to automation. To date, we have demonstrated that we can measure expression of all seven globin transcripts in multiwell format, and we can detect the relative induction of gamma globin expression by known inducers of fetal hemoglobin. In this proposal, we describe the optimization, validation, and automation of this assay as well as the performance of a pilot small molecule screen. In collaboration with the laboratory of Dr. Stuart Schreiber at the Broad Institute of Harvard and MIT, we propose to screen a library of small molecules enriched in FDA approved and other known bioactive compounds and a library of collated HDAC inhibitors. Upon completion of these studies, we hope to have identified a small number of compounds that activate fetal hemoglobin for further investigation. Furthermore, we hope to have validated the assay in a pilot screen in preparation for more extensive screens of small molecule libraries.